Cells synthesizing cancer or viral proteins alert the immune system by cleaving these proteins into peptide fragments which are presented at the cell surface in conjunction with the major histocompatibility complex class I glycoprotein (MHC-I). A deeper understanding of the mechanism by which these protein epitopes are selected will have important applications in vaccine design, and therapies against cancer, viral infections, and autoimmune diseases. In the proposed studies, the mechanism by which specific protein epitopes are selected for presentation will be investigated. Fluorescent indicators of peptide cleavage will be developed to study the cleavage of antigenic peptides within individual, living cells. Peptides will be derivatized with two fluorescent dyes that undergo energy transfer, such that the spectrum of intact peptide and each of the cleaved fragments will be different. This will enable the changing subcellular location of each peptide species to be followed in real time within living cells. Synthetic methods will be developed which can incorporate dyes at any position in a wide range of peptide sequences, and will be suitable for use with dyes that can be imaged in living cells. These investigations will provide generally applicable tools for other areas of biology. Cleavage indicators will be designed with specific biological activities for studying antigen processing and presentation. Peptides containing an epitope which binds MHC-I molecules and a nonbinding, flanking sequence will be modified with flurophores so that trimming of the antigenic epitope can be studied. The trafficking of the epitope which binds to MHC-I will be differentiated from that of the cleaved flanking sequence. The rate of transport through specific regions of the ER and other subcellular compartments will be studied, as will the localization of peptide import and efflux. We will ask how intracellular transport and cleavage contribute to the selection of specific protein fragments for antigen presentation. Peptides which have high MHC-I affinity but are not presented will be examined, as will peptides whose flanking sequences strongly affect cell surface presentation. Nonhydrolyzable linkages will be incorporated into the peptide backbone to study the role of cleavage in transport and antigen selection.